Monounsaturated fatty acids are important components of human nutrition and have been found to have health benefits, such as lowering the risk of heart disease by reducing the “bad” (low-density lipoprotein) cholesterol while maintaining the “good” (high-density lipoprotein) cholesterol (Mattson et al. (1985) Journal of Lipid Research 26:194–202). The most common naturally occurring monounsaturated fatty acid is oleic acid (18:1Δ9), which is found in abundance in “healthy” oils such as olive and canola. Therefore, it is desirable to produce oils that have increased ratios of monounsaturated to polyunsaturated fatty acids such as linoleic or linolenic acids (18:2, and 18:3, respectively), and the unsaturated fatty acids such as palmitic and stearic (16:0, and 18:0, respectively). Petroselinic acid (18:1Δ6) is a monounsaturated fatty acid that differs from the more common oleic acid by the position of the double bond in the hydrocarbon chain. It is believed that petroselinic acid is made via a pathway wherein acyl carrier protein (ACP)-bound palmitic acid (16:0) is converted to hexadecenoic acid (16:1Δ4) by the enzyme Δ4-16:0-acyl carrier protein desaturase (Δ4-16:0-ACP desaturase) (Cahoon et al. (1992) Proc Natl Acad Sci 89:11184–11188). The hexadecenoic acid is then elongated to form petroselinic acid by a β-keto-acyl-ACP synthase (KASII). Introduction of a coriander Δ4-16:0-acyl carrier protein desaturase into transgenic tobacco resulted in production of both petroselinic acid and hexadecenoic acid, neither of which is normally found in tobacco (Cahoon et al. (1992) Proc Natl Acad Sci 89:11184–11188). It is believed that specialized isoforms of ACP may be involved in this pathway which may facilitate the accumulation of petroselinic acid versus hexadecenoic acid (Suh et al. (1999) Plant J 17:679–688). Thus, it is possible to direct the accumulation of novel monounsaturated fatty acids in higher plants.
Another important product of this pathway comes from the breakdown products of the petroselinic acid and hexadecenoic acid. Chemical cleavage of the double bonds in these oils will release 12-carbon lauric acid, a component of detergents and surfactants. The other product of the cleavage of petroselinic acid is the 6-carbon adipic acid which is a monomeric component of nylon 66. Fatty acids such as petroselinic can also serve as substrates for epoxidases, hydroxylases, and other modifying activities that will produce novel products.
In a sixth embodiment, the invention also relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated suitable host cell with a chimeric gene or isolated polynucleotide of the present invention.
In a seventh embodiment, the invention concerns a Δ4-16:0-ACP desaturase polypeptide of at least 361 amino acids having at least 75% identity based on the Clustal method of alignment compared to a polypeptide selected from SEQ ID NO:2, and an ACP isoform polypeptide of at least 114 amino acids comprising at least 75% identity based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:4, and 6.
In an eighth embodiment, the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a Δ4-16:0-ACP desaturase and an ACP isoform polypeptide or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level of the Δ4-16:0-ACP desaturase and an ACP isoform polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of the Δ4-16:0-ACP desaturase and an ACP isoform polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of the Δ4-16:0-ACP desaturase and an ACP isoform polypeptide or enzyme activity in the host cell that does not contain the isolated polynucleotide.
In a ninth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a Δ4-16:0-ACP desaturase and an ACP isoform polypeptide, preferably a plant Δ4-16:0-ACP desaturase and an ACP isoform polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 300 (preferably at least 400; most preferably at least 600) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, and 5, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a Δ4-16:0-ACP desaturase and an ACP isoform amino acid sequence.
In a tenth embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a Δ4-16:0-ACP desaturase and an ACP isoform polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
In an eleventh embodiment, this invention concerns a composition, such as a hybridization mixture, comprising an isolated polynucleotide of the present invention.
In a twelfth embodiment, this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the Δ4-16:0-ACP desaturase and an ACP isoform polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.
In a thirteenth embodiment, this invention relates to a method of altering the level of expression of genes involved in petroselinic acid biosynthesis in a host cell comprising: (a) transforming a host cell with a chimeric gene of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the genes involved in petroselinic acid biosynthesis in the transformed host cell.
In a fourteenth embodiment, this invention concerns a method for producing petroselinic acid in a plant which comprises: (a) transforming a plant with a chimeric gene comprising an isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme associated with the production of petroselinic acid wherein said nucleic acid fragment encoding a polypeptide that is at least 75% identical to a polypeptide encoded by any of the nucleotide sequences set forth in SEQ ID NOs: 1 or 5, or a functionally equivalent subfragment thereof or a complement thereof operably linked to suitable regulatory sequences; (b) growing the transformed plant under conditions suitable for the expression of the chimeric gene; and (c) selecting those transformed plants producing petroselinic acid.
In fifteenth embodiment, this invention concerns a method for producing seed oil containing fatty acids having petroselinic acid in the seeds of plants which comprises: (a) transforming a plant with a chimeric gene comprising an isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme associated with the production of petroselinic acid wherein said nucleic acid fragment encoding a polypeptide that is at least 75% identical to a polypeptide encoded by any of the nucleotide sequences set forth in SEQ ID NOs: 1 or 5, or a functionally equivalent subfragment thereof or a complement thereof operably linked to suitable regulatory sequences; (b) growing a fertile mature plant from the transformed plant cell of step (a); (c) screening progeny seeds from the fertile plants of step (b) for altered levels of petroselinic acid; and (d) processing the progeny seed of step (c) to obtain seed oil containing petroselinic acid.